This is a repository copy of Living off the land : Terrestrial-based diet and dairying in the farming communities of the Neolithic Balkans. White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/164722/ Version: Published Version Article: Stojanovski, Darko, Živaljević, Ivana, Dimitrijević, Vesna et al. (16 more authors) (2020) Living off the land : Terrestrial-based diet and dairying in the farming communities of the Neolithic Balkans. PLoS ONE. e0237608. e0237608. ISSN 1932-6203 https://doi.org/10.1371/journal.pone.0237608 Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. 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Evershed3, Marie Balasse4, Adam Dowle5, Jessica Hendy6, Krista McGrath6, 7 6,8 1,2 3 Roman Fischer , Camilla Speller , Jelena JovanovićID , Emmanuelle Casanova , 9 10 11 12 13 Timothy Knowles , Lidija Balj , Goce Naumov , Anđelka Putica , Andrej Starović , 1,2 Sofija Stefanović a1111111111 1 BioSense Institute, University of Novi Sad, Novi Sad, Serbia, 2 Laboratory for Bioarchaeology, Faculty of a1111111111 Philosophy, University of Belgrade, Beograd, Serbia, 3 Organic Geochemistry Unit, School of Chemistry, a1111111111 University of Bristol, Bristol, United Kingdom, 4 Arche´ozoologie, arche´obotanique: Socie´te´s, Pratiques a1111111111 Environnements (AASPE), CNRS - Muse´um national d’Histoire Naturelle, Paris, France, 5 Department of a1111111111 Biology, Bioscience Technology Facility, University of York, York, United Kingdom, 6 BioArch, Department of Archaeology, University of York, York, United Kingdom, 7 Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom, 8 Department of Anthropology, University of British Columbia, Vancouver, Canada, 9 BRAMS Facility, School of Chemistry, University of Bristol, Bristol, United Kingdom, 10 Museum of Vojvodina, Novi Sad, Serbia, 11 Museum of Macedonia, Skopje, Macedonia, 12 The Town Museum of Sombor, Sombor, Serbia, 13 National Museum in Belgrade, Belgrade, Serbia OPEN ACCESS * [email protected] Citation: Stojanovski D, Zˇivaljević I, Dimitrijević V, Dunne J, Evershed RP, Balasse M, et al. (2020) Living off the land: Terrestrial-based diet and dairying in the farming communities of the Abstract Neolithic Balkans. PLoS ONE 15(8): e0237608. https://doi.org/10.1371/journal.pone.0237608 The application of biomolecular techniques to archaeological materials from the Balkans is Editor: Peter F. Biehl, University at Buffalo - The providing valuable new information on the prehistory of the region. This is especially relevant State University of New York, UNITED STATES for the study of the neolithisation process in SE Europe, which gradually affected the rest of Received: April 8, 2020 the continent. Here, to answer questions regarding diet and subsistence practices in early Accepted: July 29, 2020 farming societies in the central Balkans, we combine organic residue analyses of archaeo- logical pottery, taxonomic and isotopic study of domestic animal remains and biomolecular Published: August 20, 2020 analyses of human dental calculus. The results from the analyses of the lipid residues from Copyright: © 2020 Stojanovski et al. This is an pottery suggest that milk was processed in ceramic vessels. Dairy products were shown to open access article distributed under the terms of the Creative Commons Attribution License, which be part of the subsistence strategies of the earliest Neolithic communities in the region but permits unrestricted use, distribution, and were of varying importance in different areas of the Balkan. Conversely, milk proteins were reproduction in any medium, provided the original not detected within the dental calculus. The molecular and isotopic identification of meat, author and source are credited. dairy, plants and beeswax in the pottery lipids also provided insights into the diversity of diet Data Availability Statement: The raw data and in these early Neolithic communities, mainly based on terrestrial resources. We also present Mascot search results are available in the public the first compound-specific radiocarbon dates for the region, obtained directly from database on the MassIVE repository (ID: MSV000083853; http://massive.ucsd.edu; or absorbed organic residues extracted from pottery, identified as dairy lipids. https://massive.ucsd.edu/ProteoSAFe/dataset.jsp? task=e95a40e3cf8842ef83d85fd7f41a596a). Funding: This research is a result of the Project ‘BIRTH: Births, mothers and babies: prehistoric 1. Introduction fertility in the Balkans between 10,000-5000 BC’, funded by the European Research Council (ERC) The earliest attempts in the domestication of wild plants such as barley, lentil, einkorn and under the European Union’s Horizon 2020 research emmer wheat, and animal species (cattle, sheep and goat), were identified in Southeast PLOS ONE | https://doi.org/10.1371/journal.pone.0237608 August 20, 2020 1 / 27 PLOS ONE Living off the land: terrestrial-based diet and dairying in the farming communities of the Neolithic Balkans and innovation programme (Grant Agreement No. Anatolia and parts of the Levant, Syria, Iraq and Iran around 11 000 years ago [1–4]. This 640557; Principal Investigator: S.S.), https://erc. marked the beginnings of farming practices and the early stages of the Neolithic in the region. europa.eu/ We thank the NERC for partial funding From here, this new socio-economic system spread westward over land and sea, reaching of the MS facilities at Bristol (Contract R8/H10/63; www.chm.bris.ac.uk/lsmsf/). This research was Cyprus and Central Anatolia, and by 6500 calBC, the coastal areas of Western Turkey, Thes- also part supported by the Wellcome Trust [grant saly and Macedonia. Between c. 6500 and 6000 calBC, it spread throughout the Balkan Penin- no 108375/Z/15/Z]. The York Centre of Excellence sula and the southern parts of the Pannonian Plain [5–9]. Farming was introduced to the in Mass Spectrometry was created thanks to a Balkans as a complete economic concept and the entire peninsula was occupied by farmers major capital investment through Science City within five or six centuries, through a complex process of migration and interaction [10,11]. York, supported by Yorkshire Forward with funds from the Northern Way Initiative, and subsequent Genetic evidence, as well as the fact that the wild progenitors of most of the domesticates are support from EPSRC (EP/K039660/1; EP/ not native to Europe, suggests that the early farmer-migrants from the East brought with them M028127/1), FP7 Ideas ERC 324202 (Richard P. not only their agricultural knowledge, but also domesticated species of plants and animals Evershed) and ERC 812917 (Richard P. Evershed). [3,4,12–15]. The funders had no role in study design, data The Balkan Peninsula, however, is a landmass with highly diverse topography, climate and collection and analysis, decision to publish, or environment. Here, for the first time, the early farmers would have met climatic challenges preparation of the manuscript. forcing them to adapt and modify their practices and the suite of domesticates they used Competing interests: The authors have declared [16,17]. This must have affected the subsistence patterns as agricultural products were the that no competing interests exist. main source of food. The introduction of modern genetic, chemical and isotopic approaches in archaeology provided the means for gaining unprecedented insights into the human-envi- ronment relationship and, among other things, ancient human diet [10,18–22]. There is a growing body of evidence that the intensification of dairying in the Balkans followed a latitudi- nal direction [16–18,20] and the increased consumption of dairy products in sub-Continental and Continental environments of the north-central Balkans has been suggested as an attempt to supplement the losses suffered by adapting agricultural practices [16]. This specific part of the peninsula, however, remains underrepresented in bioarchaeological studies, in comparison to the Aegean coasts and Anatolia on one side, and the central and western Europe on the other. For this reason, we investigate the diet of the first farmers from a wider selection of sites from central and northern Balkans (Fig 1). More specifically, we consider the importance of milk and dairying practices and the diversity of the subsistence pattern. We combine data from molecular and stable carbon isotope analyses of organic residues from pottery, archaeo- zoological analyses and cattle mortality profiles, stable isotope analysis, i.e. nitrogen (Δ15N) and carbon (Δ13C), of cattle dentine collagen and metaproteomic analyses of human dental calculus. The δ13C isotopic analyses of lipids preserved in the pottery matrix provide direct insights into the cooking practices